Bone marrow stromal cell‐derived exosomes improve oxidative stress and pyroptosis in doxorubicin‐induced myocardial injury in vitro by regulating the transcription of GSDMD through the PI3K‐AKT‐Foxo1 pathway

Figure 1. The extraction and identification of BMSCs. (A) Ordinary optical microscope observations of BMSC morphology (×100). (B) Ordinary optical microscope analyses of adipogenic and osteogenic differentiation in BMSCs in vitro (×200). (C) Flow cytometer measurement of surface antigens (positive: CD29, CD90, and CD44; negative: CD45 and CD34). (D) Transmission electron microscope observations of BMSC‐Exos. (E) NTA analyses of BMSC‐Exo size. (F) Western blot examination of the expression of Exo surface marker proteins Alix, CD63, GM130, cytochrome c, and calnexin. (G) Laser scanning microscope evaluation of Exo uptake in H9C2 cells. BMSC, bone marrow stromal cells; Exo, exosome; NTA, nanoparticle tracking analysis.

Figure 2. BMSC‐Exos improves OS and mitochondrial damage of DOX‐treated H9C2 cells. (A) CCK‐8 assay to test viability of H9C2 cells 24 h after treatments of different concentrations of DOX (0–20 μM). (B) LDH release of H9C2 cells 24 h after treatment of DOX at above concentrations. (C) CCK‐8 assay to measure changes of cell viability after treatment of 1 μM DOX. (D) LDH release of each group. (E, F) DCFH‐DA staining to test ROS production of cells. (G, H) JC‐1 immunofluorescence to measure the changes of mitochondrial membrane potentials of cells. ***p < .001 versus the control group or the 0 μM group; ## p < .01; ### p < .001 versus the DOX + BMSC group. One‐way analysis of variance was adopted to confirm p values, with Tukey's test for post hoc multiple comparisons. Each experiment was independently repeated thrice. BMSC, bone marrow stromal cells; CCK, cell counting kit; DCFH‐DA, 20,70‐dichlorofluorescein diacetate; DOX, doxorubicin; Exo, exosome; LDH, lactic dehydrogenase; OS, oxidative stress; ROS, reactive oxygen species.

Figure 3. BMSC‐Exos ameliorates DOX‐induced H9C2 cell pyroptosis. (A, B) Western blot detection of the protein expression of NLRP3, ASC, cleaved caspase‐1, cleaved IL‐1β, cleaved IL‐18, and NT‐GSDMD in cells. (C, D) ELISA to test IL‐18 and IL‐1β levels in cell supernatants. (E, F) Immunofluorescence to examine the positive rates of NLRP3 and caspase‐1. ***p < .001 versus the control group; ## p < .01; ### p < .001 versus the DOX + BMSC group. One‐way analysis of variance was adopted to confirm p values, with Tukey's test for post hoc multiple comparisons. Each experiment was independently repeated thrice. ASC, apoptosis‐associated speck‐like protein containing A CARD; BMSC, bone marrow stromal cells; DOX, doxorubicin; ELISA, enzyme‐linked immunosorbent assay; Exo, exosome; IL, interleukin; NLRP3, nucleotide‐binding oligomerization domain, leucine rich repeat and pyrin domain containing 3; NT‐GSDMD, active N‐terminal fragment of gasdermin D.

Figure 4. BMSC‐Exos regulates GSDMD transcription through the PI3K‐AKT‐Foxo1 pathway in DOX‐induced H9C2 cells. (A) Western blot to examine the expression of p‐PI3K, p‐AKT, total‐AKT, and p‐mTOR in cells. *p < .05; **p < .01; ***p < .001 versus the DOX + BMSC group. (B) Western blot to measure the protein expression levels of total‐Foxo1 in cell nucleus and cytoplasm. (C) Western blot to determine the expression of acetylated Foxo1, phosphorylated Foxo1, and total‐Foxo1, ***p < .001 versus the DOX + BMSC group. (D) Public database to analyze the binding site sequence of Foxo1 to GSDMD. (E) Dual‐luciferase reporter gene assay to validate binding of Foxo1 to GSDMD promoter, ***p < .001 versus the OE‐NC group. (F) ChIP assay to test the enrichment level of Foxo1 in the promoter region of GSDMD, ***p < .001 versus the anti‐IgG group. The two‐tailed test was employed to confirm p values. Except for special statements, one‐way analysis of variance was adopted to confirm p values, with Tukey's test for post hoc multiple comparisons. Each experiment was independently repeated thrice. AKT, protein kinase B; BMSC, bone marrow stromal cells; ChIP, chromatin immunoprecipitation; DOX, doxorubicin; Exo,…

Figure 5. Inactivation of the PI3K‐AKT pathway abrogates the relieving impacts of BMSC‐Exos on DOX‐induced OS and pyroptosis in H9C2 cells. (A, B) DCFH‐DA staining to measure ROS levels in cells. (C, D) JC‐1 immunofluorescence to monitor the changes of potential difference of mitochondrial membrane in cells. (E, F) Western blot to test the expression of NLRP3, ASC, cleaved caspase‐1, NT‐GSDMD, cleaved IL‐1β, and cleaved IL‐18 in cells. (G, H) Western blot to measure the phosphorylation levels and total protein levels of Foxo1 in cell nucleus and cytoplasm; ***p < .001 versus the control group; ## p < .01; ### p < .001 versus the DOX group; & p < .05; && p < .01; &&& p < .001 versus the DOX + BMSC‐Exo group. One‐way analysis of variance was adopted to confirm p values, with Tukey's test for post hoc multiple comparisons. Each experiment was independently repeated thrice. AKT, protein kinase B; ASC, apoptosis‐associated speck‐like protein containing A CARD; BMSC, bone marrow stromal cells; DCFH‐DA, 20,70‐dichlorofluorescein diacetate; DOX, doxorubicin; Exo, exosome; Foxo1, forkhead box O1; GSDMD, gasdermin D; IL, interleukin; NLRP3, nucleotide‐binding oligomerization domain, leucine rich…